Involvement of the Meto/Msr System in Two Acer Species That Display Contrasting Characteristics During Germination

International Journal of Molecular Sciences

Article Involvement of the MetO/Msr System in Two Acer Species That Display Contrasting Characteristics during Germination

Natalia Wojciechowska 1,2 , Shirin Alipour 1 , Ewelina Stolarska 1, Karolina Bilska 1, Pascal Rey 3 and Ewa M. Kalemba 1,*

1 Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland; [email protected] (N.W.); [email protected] (S.A.); [email protected] (E.S.); [email protected] (K.B.) 2 Department of General Botany, Institute of Experimental Biology, Faculty of Biology, Adam Mickiewicz University, Uniwersytetu Poznańskiego6, 61-614 Poznań,Poland 3 Plant Protective Proteins (PPV) Team, Centre National de la Recherche Scientifique (CNRS), Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA), Biosciences and Biotechnology Institute of Aix-Marseille (BIAM), Aix Marseille University (AMU), 13108 Saint Paul-Lez-Durance, France; [email protected] * Correspondence: [email protected]; Tel.: +48-61-8170033

Received: 7 October 2020; Accepted: 1 December 2020; Published: 2 December 2020

Abstract: The levels of methionine sulfoxide (MetO) and the abundances of methionine sulfoxide reductases (Msrs) were reported as important for the desiccation tolerance of Acer seeds. To determine whether the MetO/Msrs system is related to reactive oxygen species (ROS) and involved in the regulation of germination in orthodox and recalcitrant seeds, Norway maple and sycamore were investigated. Changes in water content, MetO content, the abundance of MsrB1 and MsrB2 in relation to ROS content and the activity of reductases depending on nicotinamide adenine dinucleotides were monitored. Acer seeds diﬀered in germination speed—substantially higher in sycamore—hydration dynamics, levels of hydrogen peroxide, superoxide anion radicals (O ) and hydroxyl radicals ( OH), 2•− • which exhibited peaks at diﬀerent stages of germination. The MetO level dynamically changed, particularly in sycamore embryonic axes, where it was positively correlated with the levels of O2•− and the abundance of MsrB1 and negatively with the levels of OH and the abundance of MsrB2. • The MsrB2 abundance increased upon sycamore germination; in contrast, it markedly decreased in Norway maple. We propose that the ROS–MetO–Msr redox system, allowing balanced Met redox homeostasis, participates in the germination process in sycamore, which is characterized by a much higher speed compared to Norway maple.

Keywords: methionine oxidation; nicotinamide adenine dinucleotide phosphate; redox posttranslational modification; reactive oxygen species; methionine sulfoxide reductase; seeds

1. Introduction Seeds are evolutionarily important structures enabling plant reproduction. Seed germination and successful seedling establishment allow the installation of the next generation of plants. Seed germination is a complex physiological trait that can be prevented or delayed by dormancy [1–3]. The model of seed germination consists of three phases: seed imbibition, which is manifested by water uptake (ﬁrst phase), re-initiation of metabolic processes (second phase) and postgermination growth (third phase), which refers to a further increase in water uptake that results in embryo expansion [1]. Germination sensu stricto involves ﬁrst and second phases [1–3]. The molecular basis of the changes

Int. J. Mol. Sci. 2020, 21, 9197; doi:10.3390/ijms21239197 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 9197 2 of 21 in morphology, structure, metabolism, hormones and gene expression during seed germination is well characterized in Arabidopsis [4,5]. Among phytohormones, abscisic acid (ABA), giberrellins (GAs) and ethylene (ET) primarily regulate seed dormancy and germination in dicot species [6,7], notably through interplay with reactive oxygen species (ROS) [8,9]. Many studies confirmed that hormonal regulation of germination has effects at the transcriptional and proteome levels [10,11] and eventually results in global changes in enzyme activity [12]. Global interactions exist also at the transcriptome level in germinating seeds [13]. Dry mature seeds can accumulate over 10,000 mRNAs intended for the synthesis of proteins associated with redox regulation, glycolysis and protein metabolism [14]. Importantly, the activity of many enzymes is redox-regulated [12], and their oxidized forms in mature dry seeds are reduced during imbibition for metabolism restoration [15]. In this context, redox posttranslational control of seed germination has emerged as an extremely important process [14,16]. ROS are assumed to be signaling molecules, and the interplay between ROS-related transduction pathways and others, such as hormone-related pathways, is well described [17–21]. Among ROS, the signaling role of hydrogen peroxide (H2O2) is widely studied, particularly in plant acclimation to stress and during growth and development [22–24]. ROS are signaling molecules in seeds that modulate dormancy disruption and germination [25,26] and are involved in endosperm deterioration and seed reserve mobilization [27]. Three ROS, H2O2, superoxide anion radicals (O2•−) and hydroxyl radicals ( OH), have been implicated in germination, particularly in root development [28–30]. H O and • 2 2 O2•− were demonstrated to be associated with cell differentiation and proliferation, respectively [30]. Postgerminative axis growth involves O2•− production [31–33]. Less attention has been paid to the role of the short-lived but most reactive ROS, OH [34]. Indeed, OH is involved in germination [35–37]. • • More and more evidences indicate that OH could be targeted to play a role in plant cell wall loosening • thus placing these reactive molecules as important components involved in plenty of developmental processes [38]. Miller [39] and Fry [38] showed that cell-wall polysaccharides such as pectin and xyloglucan can be broken down by OH. Further analyses confirmed that OH production in the • • apoplast causes scission of specific cell wall polysaccharides in elongating maize coleoptiles as well as in the radicles and endosperm caps of germinating cress seeds [35]. ROS are thus signaling molecules involved in the regulation of seed germination [25]. Nucleic acids, especially RNA, and proteins are the most sensitive molecules to oxidation [40]. Targeted mRNA oxidation can fine-tune the cell signaling pathway that controls germination via selective translation [41]. Massive protein oxidation occurs during germination [42,43]. Importantly, selective oxidation of both mRNA and proteins is necessary for completing germination [25,27,41,44]. ROS damage to proteins can be irreversible and irreparable (i.e., carbonylation) [45]. Reversible protein oxidative modifications involve cysteine (Cys) and methionine (Met), which are the two amino acids the most prone to oxidation by ROS [46]. Oxidation of Met to methionine sulfoxide (MetO) is reversed via the action of enzymes termed methionine sulfoxide reductases (Msrs), including several isoforms that are classified as one of two types, A and B [36,37,47–51]. The Met-MetO-Met transition in proteins is considered a redox switch-regulating activity [48] in relation to ROS-initiated signaling. The reversibility of the Met redox status depends on the activity of the Msrs system, which in turn depends on the presence of redoxins and reducing agents that can regenerate Msrs [37,52]. Met metabolism was demonstrated to be essential to seed germination [16]. More precisely, proteins associated with Met synthesis and the recycling pathway [10] were identified as important for dormancy disruption [53]. However, Met redox homeostasis has never been investigated in this context. Norway maple (Acer platanoides L.) and sycamore (Acer pseudoplatanus L.) belong to the same genus but produce desiccation-tolerant and desiccation-sensitive seeds, respectively. The comparison of Norway maple and sycamore seeds became a model for studying differences between orthodox and recalcitrant seeds at important transitions, such as development [54,55], dormancy acquisition [53,56] and drying/desiccation [57–61]. However, the germination process was studied uniquely in the two species in the context of nuclear replication activity [62] and hormonal regulation [11]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 21

uniquely in the two species in the context of nuclear replication activity [62] and hormonal regulation [11]. Int. J. Mol.The Sci. seeds2020 ,of21 ,these 9197 two contrasting Acer species were characterized by distinct levels of 3ROS, of 21 MetO, MsrB1 and MsrB2 and differences in the activity of NADPH‐dependent reductases during the developmentThe seeds and of thesematuration two contrasting phases [63]Acer as wellspecies as during were characterized dehydration byand distinct desiccation levels [64]. of ROS, The MetO,MetO/Msr MsrB1 system and MsrB2was assumed and diff erencesto be involved in the activity in the ofestablishment NADPH-dependent of desiccation reductases tolerance during in theorthodox development Norway and maple maturation seeds [64]. phases Thus, [63 it] was as well necessary as during to determine dehydration whether and desiccation the MetO/Msr [64]. Thesystem MetO participates/Msr system in wasthe regulation assumed to of be germination, involved in theduring establishment which Norway of desiccation maple and tolerance sycamore in orthodoxseeds cease Norway to be mapledifferent seeds in [terms64]. Thus, of desiccation it was necessary tolerance to determine [1,65]. Of whether note, accumulation the MetO/Msr of system MsrB participateswas reported in theto be regulation related to of re germination,‐establishment during of desiccation which Norway tolerance maple in and germinating sycamore seeds seeds cease [66], tosuggesting be different that in MsrBs terms ofmight desiccation contribute tolerance to proper [1,65 germination.]. Of note, accumulation Therefore, we of MsrBinvestigated was reported whether to bethese related contrasted to re-establishment Acer seeds also of desiccation exhibit distinct tolerance features in germinating in terms of seedsROS levels, [66], suggesting MetO content, that MsrBs MsrB mightabundance contribute and the to properactivity germination. of NAD(P)H Therefore,‐dependent we reductases investigated in relation whether with these their contrasted germinationAcer seedscharacteristics. also exhibit distinct features in terms of ROS levels, MetO content, MsrB abundance and the activity of NAD(P)H-dependent reductases in relation with their germination characteristics. 2. Results 2. Results 2.1. Germination Patterns and Water Status 2.1. Germination Patterns and Water Status We first investigated the germination characteristics in Norway maple and sycamore seeds (FigureWe 1). first Norway investigated maple the embryonic germination axes characteristicsstarted to protrude in Norway at the maple12th week and sycamoreafter imbibition seeds (Figure(WAI),1 and). Norway over 5 maple weeks, embryonic over 90% axes of seeds started passed to protrude into the at thepostgerminative 12th week after growth imbibition phase. (WAI), The andgermination over 5 weeks, capacity over of 90% the ofNorway seeds passed maple intoseedlot the reached postgerminative 95 ± 2.3%. growth The dynamics phase. The of germination capacitywere strikingly of the Norway different maple in sycamore seedlot reachedseeds. Radicle 95 2.3%. protrusion The dynamics began earlier of germination at the 9–10th were week, strikingly and ± dithreefferent weeks in sycamore later, over seeds. 90% Radicleof seeds protrusion exhibited beganvisible earlier radicles. at theThe 9–10th final germination week, and three capacity weeks of later,sycamore over seeds 90% of was seeds high exhibited and reached visible 97 radicles.± 2.2%. In The this final context, germination both types capacity of Acer of seeds sycamore were highly seeds wasviable; high however, and reached Norway 97 maple2.2%. required In this context,a longer bothtime types (3 weeks) of Acer to seedsinitiate were the highlyprotrusion viable; of ± however,radicles. The Norway germination maple required speed index a longer (GSI) time confirmed (3 weeks) that to sycamore initiate the seeds protrusion accomplished of radicles. the Thegermination germination process speed faster index (GSI (GSI) = 6.72 confirmed ± 0.16) than that seeds sycamore of Norway seeds maple accomplished (GSI = 4.41 the ± germination0.14). process faster (GSI = 6.72 0.16) than seeds of Norway maple (GSI = 4.41 0.14). ± ±

FigureFigure 1. 1.Germination Germination curves curves representing representing Norway Norway maple andmaple sycamore and sycamore seeds subjected seeds to subjected germination. to Data are the means of four independent replicates the standard deviation. germination. Data are the means of four independent± replicates ± the standard deviation. During germination, the water status of Norway maple seeds exhibited a pattern similar to the typical scheme observed in many species (Figure2A). Imbibition caused a spectacular increase in water content (WC), particularly in Norway embryonic axes, whereas the WC of cotyledons was 5–10% lower. Then, the WC remained relatively stable throughout nine WAI. A subsequent peak in Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 21

During germination, the water status of Norway maple seeds exhibited a pattern similar to the

Int.typical J. Mol. scheme Sci. 2020 ,observed21, 9197 in many species (Figure 2A). Imbibition caused a spectacular increase4 of in 21 water content (WC), particularly in Norway embryonic axes, whereas the WC of cotyledons was 5–10% lower. Then, the WC remained relatively stable throughout nine WAI. A subsequent peak in tissue hydrationhydration was was reported reported in embryonicin embryonic axes ataxes the 12that the WAI, 12th while WAI, germination while germination was manifested was bymanifested radicle protrusion, by radicle and protrusion, an almost and 5% an WC almost increase 5% was WC reported increase in was cotyledons. reported Sycamore in cotyledons. seeds absorbedSycamore waterseeds absorbed in a very water distinct in a manner very distinct (Figure manner2B). The (Figure increase 2B). inThe WC increase was more in WC linear was more and dependedlinear and on depended the germination on the phase germination duration phase (R2 = 0.86). duration Sycamore (R2 = embryonic0.86). Sycamore axes deﬁnitely embryonic achieved axes adefinitely higher WC achieved at the 10th a higher WAI, whileWC at the the elongation 10th WAI, of radicles while the appeared. elongation To summarize, of radicles Norway appeared. maple To andsummarize, sycamore Norway seeds exhibited maple and entirely sycamore diﬀerent seeds hydration exhibited patterns entirely during different germination. hydration Remarkably, patterns theduring sycamore germination. seeds, whichRemarkably, displayed the fastsycamore germination seeds, comparedwhich displayed to Norway fast maplegermination seeds, compared were also characterizedto Norway maple by a seeds, very progressive were also characterized increase in water by a content.very progressive increase in water content.

Figure 2. Water sorption isoterms displaying the hydration of the embryonic axes and cotyledons of Norway maplemaple ( A(A)) and and sycamore sycamore (B )(B dry) dry seeds seeds and and during during germination. germination. Abbreviations: Abbreviations: D, Dry D, seeds; Dry I,seeds; Imbibed I, Imbibed seeds; G,seeds; Germinated G, Germinated seeds. seeds. Data Data are the are means the means of three of three independent independentreplicates replicatesthe ± ± standardthe standard deviation. deviation. Identical Identical letters letters indicate indicate groups groups not not signiﬁcantly significantly didifferentiatedﬀerentiated accordingaccording to Tukey’s test.test.

2.2. ROS Levels in Germinating Acer Seeds ROS release levels were assayed in Acer seeds during germination, as ROS are well recognized eeffectorsffectors in in redox redox signaling signaling during during this this developmental developmental stage stage [25–27 [25–27].]. Further, Further, they also they modulate also modulate protein redoxprotein status, redox particularly status, particularly in Cys and in Cys Met and residues Met residues [46]. In Norway [46]. In Norway maple seeds, maple imbibition seeds, imbibition resulted inresulted the doubling in the doubling of H2O 2ofrelease; H2O2 release; however, however, at the at 3rd the WAI, 3rd WAI, a decrease a decrease was recordedwas recorded (Figure (Figure3A). Three3A). Three weeks weeks later, later, H2O H2 2releaseO2 release doubled doubled and and then then decreased decreased at at the the 9th 9th WAI WAI and and peaked peaked againagain in germinating seeds,seeds, where where the the highest highest level level was was recorded. recorded. Imbibition Imbibition caused caused an increase an increase in O2•− inlevels O2•− inlevels Norway in Norway maple maple seeds (Figure seeds (Figure3B). After 3B). that, After O 2that,•− levels O2•− levels decreased decreased up to theup 6thto the WAI. 6th Three WAI. weeksThree later,weeks the later, level the strongly level strongly increased, increased, reaching reaching three- and three five-fold‐ and higherfive‐fold levels higher than levels that inthan dry that seeds in anddry inseeds seeds and at in the seeds 6th WAI, at the respectively. 6th WAI, respectively. Interestingly, Interestingly, imbibition resulted imbibition in a resulted 50% decrease in a 50% in release decrease of OH (Figure3C). Then, OH levels increased and remained stable up to germination. •in release of •OH (Figure• 3C). Then, •OH levels increased and remained stable up to germination. Similarly to Norway maple, imbibition of sycamore seeds was associated with an increase in the H2O22 releaserelease level level (Figure (Figure 3D).3D). Then, this level declined, increased at the 4th WAI andand declineddeclined again again two weeksweeks later. later. The The highest highest levels levels of of H 2HO22Owere2 were detected detected at theat the two two final final stages stages and and were were twice twice as high as ashigh those as inthose dry seeds.in dry In seeds. contrast In tocontrast Norway to maple Norway seeds, maple imbibition seeds, halvedimbibition the levels halved of releasedthe levels O2 •−of inreleased sycamore O2•− seeds in sycamore (Figure3 E).seeds A peak (Figure in O 3E).2•− Arelease peak wasin O measured2•− release at was the measured 6th WAI, after at the which 6th WAI, O2•− levels progressively•− decreased. No great change was observed regarding OH content in sycamore after which O2 levels progressively decreased. No great change was observed• regarding •OH exceptcontent the in sycamore somewhat except higher the levels somewhat at the 2nd higher and levels 8th WAI at the (Figure 2nd 3andF). 8th WAI (Figure 3F).

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Figure 3. A B FigureMeasurements 3. Measurements of ROS of ROS release release levels. levels. ( ) ( HydrogenA) Hydrogen peroxide peroxide (H2 O(H2)2O,(2),) ( superoxideB) superoxide anion anion radicals (O )• and− (C) hydroxyl radicals ( OH) in dry and germinating Norway maple (A–C) and radicals 2(O•−2 ) and (C) hydroxyl radicals• (•OH) in dry and germinating Norway maple (A–C) and sycamore (D–F) seeds as revealed by histochemical detection of H O and O , which were•− visualized sycamore (D–F) seeds as revealed by histochemical detection2 2 of H2•−2O2 and O2 , which were as brownvisualized staining as brown and dark staining blue staining, and dark respectively. blue staining, Abbreviations: respectively. D, Abbreviations: Dry seeds; I, Imbibed D, Dry seeds;seeds; I, G, Germinated seeds. Data are the means of six independent replicates the standard deviation. Imbibed seeds; G, Germinated seeds. Data are the means of six independent± replicates ± the standard Identicaldeviation. letters Identical indicate letters groups indicate without groups significant without diff significanterences according differences to Tukey’s according test. to Tukey’s test. Histochemical detection of hydrogen peroxide and superoxide anion radicals was performed to Histochemical detection of hydrogen peroxide and superoxide anion radicals was performed to determine the seed parts, in which ROS present. ROS were detected in embryonic axes and cotyledons determine the seed parts, in which ROS present. ROS were detected in embryonic axes and of both species (Figure3). Intense staining revealing the presence of H 2O2 was detected at the 6th WAI cotyledons of both species (Figure 3). Intense staining revealing the presence of H2O2 was detected at in Norway maple seeds, whereas a strong signal of O2•− was reported at the tip of embryonic axes at the 6th WAI in Norway maple seeds, whereas a strong signal of O2•− was reported at the tip of 9–12th WAI. Sycamore seeds exhibited more intense H2O2 staining, particularly at the germinating stage. embryonic axes at 9–12th WAI. Sycamore seeds exhibited more intense H2O2 staining, particularly at Most interestingly, all these observations corresponded to biochemical measurements. To conclude, the germinating stage. Most interestingly, all these observations corresponded to biochemical similar dynamics in the changes in H2O2 levels were reported in both types of Acer seeds, with higher measurements. To conclude, similar dynamics in the changes in H2O2 levels were reported in both concentrations in sycamore, whereas very different patterns were observed for the two other ROS, types of Acer seeds, with higher concentrations in sycamore, whereas very different patterns were which were less abundant in sycamore, particularly O2•−. observed for the two other ROS, which were less abundant in sycamore, particularly O2•−. 2.3. MetO Content in Germinating Acer Seeds 2.3. MetO Content in Germinating Acer Seeds ROS induce oxidation of proteins, particularly at the level of sulfur-containing amino acids, such as Met. Peptide-boundROS induce MetOoxidation levels of were proteins, measured particularly to determine at the whether level of they sulfur follow‐containing the changes amino in ROS acids, levels.such Initially, as Met. sycamore Peptide‐bound dry seeds MetO displayed levels were higher measured MetO content to determine than Norway whether maple they desiccated follow the oneschanges (31% andin ROS 26%, levels. respectively) Initially, (Figure sycamore4). dry Interestingly, seeds displayed at the endhigher of theMetO germination content than process, Norway seedsmaple of both desiccated species containedones (31% similar and 26%, MetO respectively) content; however, (Figure the dynamics4). Interestingly, of MetO at changes the end during of the germinationgermination stages process, were entirelyseeds of di bothfferent. species Except contained in Norway similar maple MetO cotyledons, content; imbibitionhowever, the resulted dynamics in a significantof MetO changes decrease during in MetO germination levels, and lowstages levels were were entirely maintained different. for theExcept first twoin Norway weeks after maple imbibition.cotyledons, Remarkably, imbibition the resulted sycamore in embryonic a significant axes decrease exhibited in much MetO lower levels, MetO and contents low (~14%)levels were at maintained for the first two weeks after imbibition. Remarkably, the sycamore embryonic axes exhibited much lower MetO contents (~14%) at 2nd and 8th WAI. Of note, the embryonic axes of

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2ndboth and Acer 8th species WAI. displayed Of note, the a peak embryonic in the axesMetO of level both atAcer the species6th WAI. displayed In conclusion, a peak our in the data MetO revealed level atmuch the 6thmore WAI. pronounced In conclusion, variations our data in MetO revealed level much in sycamore more pronounced compared variationsto those in in Norway MetO level maple, in sycamoreparticularly compared in embryonic to those axes. in Norway maple, particularly in embryonic axes.

Figure 4. Measurement of protein protein-bound‐bound methionine sulfoxide (MetO) levels in dry and germinating Norway maplemaple and and sycamore sycamore seeds. seeds. Abbreviations: Abbreviations: D, DryD, Dry seeds; seeds; I, Imbibed I, Imbibed seeds; seeds; G, Germinated G, Germinated seeds. Dataseeds. are Data the are means the ofmeans three of independent three independent replicates replicatesthe standard ± the standard deviation. deviation. Identical Identical letters indicate letters ± groupsindicate not groups significantly not significantly differentiated differentiated according according to Tukey’s to test. Tukey’s test. 2.4. MsrB1 and MsrB2 Abundance in Germinating Acer Seeds 2.4. MsrB1 and MsrB2 Abundance in Germinating Acer Seeds Oxidation of Met to MetO is reversible thanks to the action of Msr enzymes. Two isoforms related Oxidation of Met to MetO is reversible thanks to the action of Msr enzymes. Two isoforms to type-B, B1 and B2, have been previously reported to fulfill essential roles in seed development or related to type‐B, B1 and B2, have been previously reported to fulfill essential roles in seed longevity [64,67]. We thus investigated the abundance of these two enzymes using Western blotting development or longevity [64,67]. We thus investigated the abundance of these two enzymes using analysis during the germination process in Acer seeds (Figures5 and6). In contrast to Norway maple, Western blotting analysis during the germination process in Acer seeds (Figures 5 and 6). In contrast both isoforms were detected in sycamore. In the embryonic axes, MsrB1 was detected in all of the to Norway maple, both isoforms were detected in sycamore. In the embryonic axes, MsrB1 was studied stages, with a varying abundance (Figure5). Imbibed sycamore displayed decreased MsrB1 detected in all of the studied stages, with a varying abundance (Figure 5). Imbibed sycamore content; a strong increase was noted at the 2nd and 6th WAI, and the protein abundance decreased displayed decreased MsrB1 content; a strong increase was noted at the 2nd and 6th WAI, and the significantly during the subsequent weeks. A relatively low protein level was detected in germinated protein abundance decreased significantly during the subsequent weeks. A relatively low protein seeds (Figure5A). In cotyledons, the level of MsrB1 in dry and imbibed seeds was similar. From the level was detected in germinated seeds (Figure 5A). In cotyledons, the level of MsrB1 in dry and 2nd to the 6th WAI, a gradual increase in MsrB1 protein level was detected, with a substantial peak imbibed seeds was similar. From the 2nd to the 6th WAI, a gradual increase in MsrB1 protein level at week 6. At week 8, the amount of protein dropped sharply, and it was almost undetectable in was detected, with a substantial peak at week 6. At week 8, the amount of protein dropped sharply, germinated seeds (Figure5B). and it was almost undetectable in germinated seeds (Figure 5B).

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FigureFigure 5. 5.Western Western blot blot analysis analysis and and densitometry densitometry analysis analysis of of methionine methionine sulfoxide sulfoxide reductase reductase (Msr)B1 (Msr)B1 proteinprotein inin thethe embryonicembryonic axesaxes (A(A)) andand cotyledonscotyledons (B(B)) ofof drydry andand germinatinggerminating sycamoresycamore seeds.seeds. Abbreviations:Abbreviations: D, D, Dry Dry seeds; seeds; I, I, Imbibed Imbibed seeds; seeds; G, G, Germinated Germinated seeds. seeds. The The data data are are the the means means of of three three independentindependent replicates replicates the± the STDs. STDs. The The same same letters letters indicate indicate groups groups that were that not were signiﬁcantly not significantly diﬀerent ± Int. J.according differentMol. Sci. 2020 according to Tukey’s, 21, x FOR to test. PEERTukey’s REVIEW test. 8 of 21

Figure 6. Western blot analysis and densitometry analysis of MsrB2 protein in the embryonic axes (A) Figure 6. Western blot analysis and densitometry analysis of MsrB2 protein in the embryonic axes and cotyledons (B) of dry and germinating Norway maple seeds and in the embryonic axes of D and (A) and cotyledons (B) of dry and germinating Norway maple seeds and in the embryonic axes of D germinating sycamore seeds (C). Abbreviations: D, Dry seeds; I, Imbibed seeds; G, Germinated seeds. and germinating sycamore seeds (C). Abbreviations: D, Dry seeds; I, Imbibed seeds; G, Germinated The data are the means of three independent replicates the STDs. The same letters indicate groups seeds. The data are the means of three independent replicates± ± the STDs. The same letters indicate that are not signiﬁcantly diﬀerent according to Tukey’s test. groups that are not significantly different according to Tukey’s test.

In sycamore embryonic axes, a relatively high level of MsrB2 abundance was detected at all studied stages (Figure 6C). In imbibed seeds, the protein amount was higher than that in dry seeds. In the last weeks of germination sensu stricto phase, the protein amount further increased, reaching the highest level in germinated seeds. Interestingly, MsrB2 displayed reverse patterns of abundance in Norway maple and sycamore seeds during germination. A gradual increase in MsrB2 abundance was detected in sycamore, whereas a gradual decrease was reported in Norway maple.

2.5. Activity of NAD(P)H‐Dependent Reductases in Germinating Acer Seeds Oxidoreductases constitute the major class of enzymes that catalyze a wide variety of redox reactions using different substrates. This class includes NADH‐ and NADPH‐dependent groups of enzymes, with a relatively small group of oxidoreductases using both cofactors [68]. The activity of NAD(P)H‐dependent reductases reflects the global cell redox status and the ability to maintain redox homeostasis via effectors such as Msrs. Importantly, the regeneration system for cytosolic and some plastidial Msrs is based on NADPH‐dependent mechanisms [69]. In other respects, the source of the reducing power of plastidial Msrs, such as MsrB2, in a nonphotosynthetic context like seeds remains unknown. When comparing both Acer species, the greatest difference in the activity of NADH‐dependent reductases was reported in dry seeds (Figure 7). At this stage, a much higher activity level was measured for NADH‐dependent reductases in Norway maple seeds compared to sycamore. Then, a strong decrease (−66%) was observed upon imbibition in both cotyledons and embryonic axes of Norway maple. It is important to note that the peaks of activity were detected at distinct stages of the seed germination process in the two species. In Norway maple embryonic axes, the activity of NADH‐dependent reductases peaked at the 3rd week. Then, relatively constant and similar activity was sustained up to the germinated stage as well as in cotyledons. In contrast, in embryonic axes of sycamore, the activity of NADH‐dependent reductases was quite similar and low up to the 4th WAI and then substantially increased and peaked at the 8th week, with a 4‐fold higher level compared to that of the initial stage. No variation was detected in sycamore cotyledons, the activity being quite low throughout the germination process. Interestingly, the activity of NADH‐dependent reductases was twice as low in sycamore cotyledons compared to that in Norway maple cotyledons.

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The MsrB2 protein was detected in embryonic axes and cotyledons of Norway maple (Figure6A,B) and in the embryonic axes of sycamore (Figure6C). A relatively high protein amount was detected in dry Norway maple embryonic axes (Figure6A). Following imbibition, the level of MsrB2 was somewhat reduced and further gradually decreased in the subsequent weeks. At the 9th WAI, the protein abundance was very low, whereas in germinated seeds, it was barely detectable (Figure6A). In cotyledons, the amount of MsrB2 detected in all studied stages was lower compared to that in embryonic axes (Figure6B). There was a slight decrease in protein content following imbibition. In the subsequent weeks, the MsrB2 abundance was higher, particularly at the 6th WAI, while in germinated seeds, the protein level was signiﬁcantly reduced (Figure6B). In germinated Norway maple seeds, the MsrB2 protein was almost undetectable. In sycamore embryonic axes, a relatively high level of MsrB2 abundance was detected at all studied stages (Figure6C). In imbibed seeds, the protein amount was higher than that in dry seeds. In the last weeks of germination sensu stricto phase, the protein amount further increased, reaching the highest level in germinated seeds. Interestingly, MsrB2 displayed reverse patterns of abundance in Norway maple and sycamore seeds during germination. A gradual increase in MsrB2 abundance was detected in sycamore, whereas a gradual decrease was reported in Norway maple.

2.5. Activity of NAD(P)H-Dependent Reductases in Germinating Acer Seeds Oxidoreductases constitute the major class of enzymes that catalyze a wide variety of redox reactions using different substrates. This class includes NADH- and NADPH-dependent groups of enzymes, with a relatively small group of oxidoreductases using both cofactors [68]. The activity of NAD(P)H-dependent reductases reflects the global cell redox status and the ability to maintain redox homeostasis via effectors such as Msrs. Importantly, the regeneration system for cytosolic and some plastidial Msrs is based on NADPH-dependent mechanisms [69]. In other respects, the source of the reducing power of plastidial Msrs, such as MsrB2, in a nonphotosynthetic context like seeds remains unknown. When comparing both Acer species, the greatest difference in the activity of NADH-dependent reductases was reported in dry seeds (Figure7). At this stage, a much higher activity level was measured for NADH-dependent reductases in Norway maple seeds compared to sycamore. Then, a strong decrease ( 66%) was observed upon imbibition in both cotyledons and embryonic axes of − Norway maple. It is important to note that the peaks of activity were detected at distinct stages of the seed germination process in the two species. In Norway maple embryonic axes, the activity of NADH-dependent reductases peaked at the 3rd week. Then, relatively constant and similar activity was sustained up to the germinated stage as well as in cotyledons. In contrast, in embryonic axes of sycamore, the activity of NADH-dependent reductases was quite similar and low up to the 4th WAI and then substantially increased and peaked at the 8th week, with a 4-fold higher level compared to that of the initial stage. No variation was detected in sycamore cotyledons, the activity being quite low throughout the germination process. Interestingly, the activity of NADH-dependent reductases was twice as low in sycamore cotyledons compared to that in Norway maple cotyledons. The activity of NADPH-dependent reductases was higher in Norway maple compared to sycamore (Figure7). In embryonic axes of Norway maple, a peak was detected in dry seeds, the activity being twice as high as that in cotyledons at this stage. Then, the activity gradually decreased in axes, while in cotyledons, a peak was observed at the 6th WAI. Sycamore seeds displayed lower activity of NAD(P)H-dependent reductases compared to Norway maple. The activity was constant in embryonic axes throughout the germination stages. Sycamore cotyledons exhibited activity that was twice as low as Norway maple activity, except in germinated seeds, where a similar level was measured in both embryonic axes and cotyledons of this species. Int. J. Mol. Sci. 2020, 21, 9197 9 of 21 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 21

Figure 7. Activity of NAD(P)H-dependent reductases in the embryonic axes and cotyledons of Norway Figure 7. Activity of NAD(P)H‐dependent reductases in the embryonic axes and cotyledons of maple and sycamore dry and germinating seeds. Abbreviations: D, Dry seeds; I, Imbibed seeds; NorwayG, Germinated maple and seeds sycamore. Statistically dry significantand germinating differences seeds. are indicated Abbreviations: with di ffD,erent Dry letters seeds; (one-way I, Imbibed seeds;ANOVA, G, Germinated followed by seeds. Tukey’s Statistically test at p 0.05). significant differences are indicated with different letters ≤ (one‐way ANOVA, followed by Tukey’s test at p ≤ 0.05). 2.6. Analysis of Correlations between Studied Parameters TheTo activity investigate of NADPH the relationship‐dependent between reductases the levels was of ROS, higher MetO in and Norway Msrs during maple germination compared to sycamorein orthodox (Figure and 7). recalcitrant In embryonicAcer seeds,axes of Pearson Norway correlation maple, a analyses peak was were detected made between in dry allseeds, the the activityparameters being twice tested as (Figure high as8). that The in MetO cotyledons content at was this correlated stage. Then, with the the activity levels gradually of ROS only decreased in sycamore seeds and was positively correlated with O release values and negatively with OH ones. in axes, while in cotyledons, a peak was observed at2•− the 6th WAI. Sycamore seeds displayed• lower activityFurthermore, of NAD(P)H in this‐dependent species, the MetOreductases content compared was negatively to Norway correlated maple. with theThe MsrB2 activity abundance was constant in in embryonicembryonic axes andthroughout positively the correlated germination with the stages. MsrB1 Sycamore abundance cotyledons in cotyledons. exhibited The abundance activity that of MsrB1 was positively correlated with the activity of NADH-dependent reductases. In Norway was twice as low as Norway maple activity, except in germinated seeds, where a similar level was maple, no significant relationship between ROS levels, MetO content and Msrs abundance could be measured in both embryonic axes and cotyledons of this species. detected. However, the MsrB2 abundance was positively correlated with the activities of NADH- and NADPH-dependent reductases in embryonic axes and of NADPH-dependent reductases in cotyledons. 2.6. Analysis of Correlations between Studied Parameters To investigate the relationship between the levels of ROS, MetO and Msrs during germination in orthodox and recalcitrant Acer seeds, Pearson correlation analyses were made between all the parameters tested (Figure 8). The MetO content was correlated with the levels of ROS only in sycamore seeds and was positively correlated with O2•− release values and negatively with •OH ones. Furthermore, in this species, the MetO content was negatively correlated with the MsrB2 abundance in embryonic axes and positively correlated with the MsrB1 abundance in cotyledons. The abundance of MsrB1 was positively correlated with the activity of NADH‐dependent reductases. In Norway maple, no significant relationship between ROS levels, MetO content and Msrs abundance could be detected. However, the MsrB2 abundance was positively correlated with the activities of NADH‐ and NADPH‐dependent reductases in embryonic axes and of NADPH‐dependent reductases in cotyledons.

Int. J. Mol. Sci. 2020, 21, 9197 10 of 21 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 21

Figure 8. Correlation matrices calculated for embryonic axes and cotyledons of Norway maple Figure 8. Correlation matrices calculated for embryonic axes and cotyledons of Norway maple and and sycamore dry and germinating seeds based on levels of hydrogen peroxide (H2O2), superoxide sycamore dry and germinating seeds based on levels of hydrogen peroxide (H2O2), superoxide anion anion radical (O2•−) and hydroxyl radical ( OH), water content (WC), levels of protein-bound •− • methionineradical (O2 sulfoxide) and hydroxyl (MetO), radical the abundance (•OH), water of methioninecontent (WC), sulfoxide levels of reductase protein‐bound isoforms methionine (MsrB1, MsrB2),sulfoxide activity (MetO), of NADH-dependentthe abundance of reductases methionine (NADH-dep. sulfoxide reductase red.), activity isoforms of NADPH-dependent (MsrB1, MsrB2), reductasesactivity of (NADPH-dep. NADH‐dependent red.). Thereductases percentage (NADH of WC‐dep. was red.), ArcSin activity transformed. of NADPH Crossed‐dependent numbers indicatereductases non-signiﬁcant (NADPH‐dep. correlation red.). The ( ppercentage> 0.05). Thof WC more was red ArcSin color thetransformed. more negative Crossed correlation, numbers whereasindicate thenon more‐significant blue the correlation more positive (p > correlation. 0.05). Th more red color the more negative correlation, whereas the more blue the more positive correlation. 3. Discussion 3. Discussion Seeds of Norway maple and sycamore were reported to diﬀer in the levels of ROS, MetO, the abundanceSeeds of Norway of MsrB1 maple and and MsrB2, sycamore and thewere activity reported of NADPH-dependentto differ in the levels reductasesof ROS, MetO, during the maturationabundance andof MsrB1 drying [and63,64 MsrB2,]. The control and the of Metactivity redox of homeostasis NADPH‐dependent by the MetO reductases/Msr/Met systemduring wasmaturation proposed and to drying accompany [63,64]. the The acquisition control of of Met desiccation redox homeostasis tolerance in by Norway the MetO/Msr/Met maple seeds [system57,64]. Aswas the proposed role of the to accompany MetO/Msr/Met the systemacquisition has of not desiccation yet been documented tolerance in inNorway seeds during maple seeds germination, [57,64]. weAs investigatedthe role of the whether MetO/Msr/Met this system system could has be involved not yet been in the documented regulation of in germination seeds during in germination, the two Acer specieswe investigated because desiccation whether this tolerance system is could lost in be orthodox involved seeds in the during regulation this developmental of germination stage in the [1] andtwo moreAcer species precisely because coincides desiccation with visible tolerance radicle is elongation lost in orthodox [70,71]. seeds Thus, during searching this for developmental possible regulatory stage functions[1] and more of the precisely MetO/Msrs coincides system with seemed visible very radicle interesting, elongation particularly [70,71]. Thus, taking searching in consideration for possible the importantregulatory role functions of Met synthesisof the MetO/Msrs and recycling system in seed seemed germination very interesting, [10,16]. particularly taking in consideration the important role of Met synthesis and recycling in seed germination [10,16].

3.1. Differential Behavior of Acer Seeds during Germination Under identical stratification conditions, sycamore seeds exhibited a higher GSI and germinated faster than Norway maple seeds (Figure 1). The time needed for the achievement of

Int. J. Mol. Sci. 2020, 21, 9197 11 of 21

3.1. Differential Behavior of Acer Seeds during Germination Under identical stratification conditions, sycamore seeds exhibited a higher GSI and germinated faster than Norway maple seeds (Figure1). The time needed for the achievement of germination phase III manifested by radicle protrusion is different in Acer species and takes 12–20 weeks for Norway maple and 8–15 weeks for sycamore [72]. Consistently, we observed that sycamore seeds began to elongate their radicles three weeks earlier than Norway maple seeds (Figure1). Cold storage was reported to eliminate dormancy in Acer and accelerate germination of Norway maple seeds [11], but here, the analyzed seeds were not stored and directly subjected to germination after harvesting. This 3-week difference could be related to the discrete hormonal balance since mature sycamore seeds contain less ABA than Norway maple seeds [73], as reflected by the degree of the physiological dormancy of the two species. A dynamic balance of two antagonistic hormones, ABA and GAs, controls seed dormancy and germination [6,7]. Consequently, ABA decreased, while GA increased the germination rate but not the germination speed in Norway maple seeds [11]. In contrast, GA substantially increased the germination rate and germination speed in sycamore seeds [53]. In particular, ABA and GA levels were reported to substantially affect the proteome of germinating Norway maple seeds [11] and sycamore seeds [53]. Additionally, the hormone balance supported by ROS homeostasis enables successive germination [74]. Particularly in imbibed seeds, H2O2 increases the level of ABA [8] and induces synthesis of ethylene [75]. Thus, ROS signals combined with GA- and ABA-related signaling transduction pathways fulfill key roles in the germination process that need to be further delineated. The progressive increase in water content observed in sycamore seeds (Figure2), which does not fit the classical triphasic seed germination model [1], might be as well related to differential behavior of Acer seeds during germination.

3.2. Relationship between ROS and MetO Levels during Germination of Sycamore Seeds ROS, including H O ,O and OH, participate in regulating the germination process in 2 2 2•− • seeds, particularly root development [25,29,30,74,76]. Bailly et al. [70] established the concept of the “oxidative window for germination” linked to a specific range of ROS levels, which leads to the initiation and further progression of seed germination. Germinated Norway maple and sycamore seeds 1 contained 3 nM and 4 nM g− DW of H2O2 (Figure3). Importantly, H 2O2 concentrations ranging from 1–10 nM are assumed to be physiological concentrations involved in signaling [77]. Thus, H2O2 levels, which are slightly higher in sycamore seeds, are within the signaling range, indicating that the peaks in ROS might result in the high germination rate recorded in both species (Figure1). H2O2 is the major ROS type that is considered a signaling molecule during seed germination [25]. H2O2 provides the optimum oxygen concentration for faster imbibition and germination [78]. The peak of H2O2 detected at the 4th and 6th WAIs in sycamore and Norway maple seeds, respectively, possibly reflects the signaling function in the early stages of dormancy disruption. This two-week delay is very likely associated with the different dynamics of germination because the time needed for initiation of radicle protrusion is longer in Norway maple [72] and lasted three weeks in our studies (Figure1). Higher H 2O2 levels together with the occurrence of peaks at earlier stages of the germination sensu stricto phase are likely to contribute to the faster germination of sycamore seeds. As a result of faster water uptake, earlier peaks of ROS were the indices of rapid germination rate of recalcitrant Avicennia marina seeds [79]. Evidently, Norway maple and sycamore display different germination-related ROS signaling patterns. A clear increase was reported in the concentrations of O2•− in Norway maple seeds at the final germination stages, which is in line with the fact that postgerminative axis growth requires O [31–33]. Interestingly, the highest OH levels appeared 2•− • just before radicle elongation uniquely in sycamore seeds. OH functions in cell wall loosening [34]; • hence, the burst of OH reported during radish germination [80] and in our studies (Figure3) can be • assumed to be an important component of the germination program. Among ROS, OH has the highest oxidative potential in Met oxidation [48], and a correlation • between the levels of OH and MetO was observed in sycamore seeds during germination (Figure8). • Int. J. Mol. Sci. 2020, 21, 9197 12 of 21

Interestingly, OH was shown to affect MetO levels in desiccated Norway maple embryonic axes [64]. • Another ROS type causing Met to oxidize is H2O2 [48]. Mature Norway maple and sycamore seeds differed in MetO content and showed distinct responses to dehydration and desiccation [64], with no change in sycamore and a decrease in the MetO level in Norway maple along with desiccation. In contrast, substantial changes in the MetO content were observed upon germination, more specifically in sycamore seeds (Figure4). Sycamore embryonic axes displayed further decreased MetO levels at the 2nd WAI (Figure4) and a peak at the 6th WAI coinciding with that of O 2•− (Figure3), as reflected by the positive correlation between the two parameters. In plants, the MetO content differs substantially depending on physiological context, organ type and very likely ROS homeostasis and scavenging capacity. For example, unstressed leaves from various species contain 2–6% MetO, and environmental constraints were reported to increase this value up to more than 50% [81–83]. The oxidation state characteristic of seeds [27] was particularly reflected by the high MetO level exceeding 30% in dried Acer seeds [64]. The dynamically changing MetO level in sycamore germinating seeds (Figure4) is consistent with the fact that controlled protein oxidation is required for successive seed germination [27]. The fine control of Met redox status in concert with ROS signals and Msr activity very likely modulates protein functions through posttranslational modification [84].

3.3. Involvement of MsrB2 in Sycamore Seed Germination MetO is easily reduced by Msr enzymes present in all living organisms [85]. It is supposed that in plants, Msr enzymes play more complex roles than in other organisms such as yeast or mammals because of the much larger number of isoforms in subcellular compartments [37,86]. In this work, we focused on the two isoforms of plastidial MsrB proteins, namely, MsrB1 and MsrB2. Initially, the functions of these proteins were considered only in photosynthetic tissues [20,36]. Currently, their presence has also been confirmed in other organs such as flowers, stems, roots and seeds [67,87,88]. Most available information about their functions relates to their contribution in responses to biotic [89] and abiotic stresses [87,90]. However, these isoforms are also involved in several developmental processes such as seed maturation, desiccation and longevity [63,64]. In sycamore seeds, both MsrB isoforms were detected, notably with an increasing level of MsrB2 in embryonic axes during germination sensu stricto phase (Figure6). In Norway maple seeds, only one isoform, MsrB2, was detected using western blot analysis as reported during seed maturation [63] and desiccation [64]. The low abundance of MsrB1 in Norway maple seeds might be compensated by MsrB2 or other Msrs including MsrA as reported by Staszak and Pawłowski [56]. The high MsrB2 abundance in sycamore seeds very likely results in enhanced Msr activity and thus reduction of oxidized proteins that could delay germination in their oxidized state [91]. At the present time, very little is known regarding targets of Msrs in plants. In Arabidopsis leaves, among MsrB1 partners, 13 plastidial and 11 nonplastidial proteins were identified [92]. They included elongation factor 2 and 26S proteasome regulatory subunit, which are very sensitive to oxidation [93,94]. Therefore, protein synthesis and turnover can be processed more efficiently, further contributing to germination speed, particularly in sycamore seeds due to the high abundance of MsrB2 in this species. Interestingly, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as an MsrB1 partner [92]. Reduced GADPH acts in glycolysis releasing NADH, whereas oxidized GADPH redirects glycolysis to the pentose phosphate pathway (PPP), allowing the cell to generate more NADPH [95]. Thus, more targets of Msrs need to be identified, particularly in seeds, to unravel the roles of these reductases during germination. Most interestingly, MsrB2 uniquely exhibited a linear increase in abundance in embryonic axes of germinating sycamore seeds (Figure6), likely indicating upregulation of MsrB2 gene expression in this species. The promoter regions of both MsrB1 and MsrB2 contain several ABA responsive elements, GA and ABA responsive elements and binding sites for GA-regulated transcription factors [96]. In this context, plant hormones, predominantly ABA and GA, which are involved in regulation of MsrBs, might be associated with control of the MetO/Msr system in germinating Acer seeds. Increasing MsrB2 abundance in sycamore embryonic axes (Figure6) seems to have a beneficial Int. J. Mol. Sci. 2020, 21, 9197 13 of 21 effect on significantly reduced MetO content during all germination stages (Figure4). Recently, methionine sulfoxidation of a nonripening transcription factor was identified as a posttranslational modification involved in regulation of tomato ripening [97]. Thus, the support of MsrBs in sycamore seeds might be beneficial for proper seed germination processes and, putatively, germination speed in this species by modulating the redox status of key actors of this developmental phase. The role of NADH- and NADPH-dependent reductases is extremely broad, and their functions are still being elucidated, particularly in seeds [68] because, for now, distinct activities of the two groups of enzymes were reported in dehydrated sycamore seeds and desiccated Norway maple seeds and assigned to contrasting desiccation tolerance of the two Acer species [57]. High activity levels were measured for NADH-dependent reductases in sycamore embryonic axes at the three final germination stages (Figure7). These enzymes may include NADH dehydrogenases used in the electron transport chain for generation of ATP, which peaked at final germination stages at a level twice high compared to the dry stage (Figure7). NADH for these reactions might originate in sycamore seeds from the activity of GAPDH, which can interact with MsrB1 [92]. Arabidopsis GADPH contains six, nine and nine Met residues in A, B and C2 subunits, respectively. Possibly, GADPH might be kept in active reduced form by MsrB1. Oxidized GAPDH generate more NADPH via pentose phosphate pathway [95]. Thus, we speculate that MsrB1 might be associated with the generation of reducing power. The regeneration system of MsrB2 involves a thioredoxin (Trx) and ferredoxin-Trx reductase-dependent mechanism, whereas the mechanism related to MsrB1 uses glutaredoxin (Grx)- or GSH/Grx-dependent mechanisms [52,98]. For MSRB1, the reducing source in seeds could involve NADPH via glutathione reductase and GSH as reported in [98]. For MsrB2, the exact reducing source is very elusive based on what is known in photosynthetic plastids. The activity of NADH-dependent reductases was correlated with the abundance of MsrB1 in sycamore seeds and MsrB2 in Norway maple seeds (Figure8). Additionally, the abundance of MsrB2 protein was positively correlated with the activity of NADPH-dependent reductases in embryonic axes and cotyledons of Norway maple seeds (Figure8) suggesting that this group of reductases might be more e fficient in Msr regeneration in this species because the activity of NADPH-dependent reductases was higher in this species compared to sycamore (Figure7). Thus, we suggest based on correlations that NADH or NADPH could be the reducing source for MsrB2 via intermediates that are presently unknown.

4. Materials and Methods

4.1. Material All experiments were performed on embryonic axes and cotyledons of seeds belonging to two species of Acer, Norway maple and sycamore. Seeds were collected at 23 and 24 weeks after ﬂowering from single trees growing in Kórnik Western Poland, 52◦24037” N, 17◦090515” E in the year 2018. Seeds were dried (D) to 10% (Norway maple) and 30% (sycamore) water content. Subsequently, seeds were hydrated for 24 h, and the imbibed seeds (I) were placed in containers on wet towels and kept at 3 ◦C. Wet towels were changed every week to avoid microorganisms growing. During the germination sensu stricto phase, the material was collected for analyses at regular intervals for each species (for Norway maple every 3 weeks (3, 6, 9), for sycamore every 2 weeks (2, 4, 6, 8)). The last stage collected for analysis constituted germinated seeds (with radicle protruding outside the seed coat) (G). Water uptake was monitored at each analyzed stage via the low-constant-temperature oven method [99]. Moisture content was measured in three lots of 20 embryonic axes and 10 cotyledons each after drying at 17 h at 103 ◦C and calculated based on fresh weight.

4.2. Germination Test Seeds were imbibed, placed in closed plastic boxes (4, 50 seeds each) and subjected to cold stratification (3 ◦C) under a 12 h light/12 h dark cycle. Cold stratification of seeds was conducted for 10–12 weeks. Seeds were assayed as germinated when the radicle protruded to 5 mm above the Int. J. Mol. Sci. 2020, 21, 9197 14 of 21 seed testa. Germination speed index (GSI) displaying a time-weighted cumulative germination that measures the speed of germination and quantifies the seedling vigor was calculated according to [100].

4.3. Determination of ROS Release

4.3.1. H2O2

The level of H2O2 release was measured according to the method described by Schopfer et al. [78]. Seeds were incubated in 1.2 mL of a solution containing 20 mM phosphate buffer (pH 6), 5 µM scopoletin 1 and 1 U mL− peroxidase. The material was incubated in darkness on a shaker at 150 rpm for 1 h at room temperature (RT). After a short centrifugation interval, the fluorescence was measured at an excitation wavelength of 346 nm and an emission wavelength of 455 nm using an Infinite M200 PRO (Tecan, Männedorf, Switzerland) plate reader and Magellan software. The results were shown in picomoles of H2O2 per gram of dry weight (DW) per hour.

4.3.2. O2•−

The release of O2•− was determined using a method described by Choi et al. [101]. Seeds were incubated in 1.2 mL of solution consisting of 50 mM phosphate buﬀer (pH 7.8), 0.05% nitro blue tetrazolium (NBT; Sigma, St. Louis, MO, USA) and 10 mM sodium azide. Incubation was performed for 30 min at room temperature in darkness. Subsequently, 750 µL of this reactive solution was heated for 30 min at 85 C, cooled and centrifuged for 1.5 min at 10,000 g RCF. The precipitate was dissolved ◦ × in dimethyl sulfoxide (DMSO) consisting of 2 M KOH by shaking for 30 min at 150 rpm and vortexing every 5 min. The level of released O2•− was measured at 719 nm using an Inﬁnite M200 PRO (Tecan, Männedorf, Switzerland) plate reader and Magellan software. The results are presented as ∆A719 values per gram of DW per hour.

4.3.3. OH • The level of released OH was determined according to the methods of Schopfer et al. [80]. • The material was incubated in 1.2 mL of a reaction mixture containing 20 mM phosphate buffer (pH 6) and 2.5 mM sodium benzoate. Incubation was performed for 3 h at RT in darkness. Moreover, the samples were constituted by shaking at 150 rpm. Then, the solution was briefly centrifuged, and the fluorescence was measured at an excitation wavelength of 305 nm and an emission wavelength of 407 nm using an Infinite M200 PRO (Tecan, Männedorf, Switzerland) plate reader and Magellan software (Tecan, Männedorf, Switzerland). The results were expressed in relative fluorescence units (RFU) per gram of DW per hour. Each analysis was performed on six replicates. For each experiment, six seeds were taken (without separating embryonic axes and cotyledons).

4.3.4. Histochemical Detection of ROS The method described by Daudi and O’Brien [102] with some modifications mentioned by Kalemba et al. [31] was used to detect H2O2. Seeds were incubated in a solution of 3,3-diaminobenzidine (DAB) prepared in sodium phosphate buffer. Seeds were incubated in DAB solution for 24 h starting with 30 min of infiltration in the vacuum pump. In the presence of H2O2, DAB is oxidized and forms an insoluble a reddish-brown polymer. The detection of O2•− was performed using a method described by Kumar et al. [103] in which material was incubated for 1 h in 0.2% nitrotetrazolium blue chloride (NBT) in sodium phosphate buffer (pH 7.5). The presence of O2•− is visualized as a dark blue color, which is an insoluble formazan dye formed in the presence of a superoxide anion. After incubation, seeds were washed 3 times in water, and subsequently images were taken using a Nikon D3100 digital camera attached to a binocular microscope. Int. J. Mol. Sci. 2020, 21, 9197 15 of 21

4.4. Determination of Peptide-Bound MetO Level Levels of MetO were determined according to the method of Baxter et al. [104] adapted to seed material [64] using an Agilent Infinity II 1260 model HPLC system (Agilent Technologies, Wilmington, DE, USA) equipped with an Agilent Poroshell 120 Stablebond Aq, 3.0 150 mm, 2.7 µm particle × column heated to 40 ◦C, mobile phases based on water (A) and potassium phosphate buffer combined with acetonitrile and isopropanol (B) and the standards of methionine (Met) and MetO with detection wavelengths of 214 nm and a reference at 590 nm. Protein digestion was performed for 20 h at 37 ◦C with a mixture of proteases including pronase E, leucine aminopeptidase and prolidase. The elution 1 scheme was 0% B from 0.0 to 5.0 min (flow rate of 0.15 mL min− ), 0 to 16% B from 5.0 to 8.0 min 1 1 (flow rate of 0.3 mL min− ), 16 to 100% B from 8.0 to 16.0 min (flow rate of 0.3 mL min− ) and 0% B 1 from 16.0 to 18.0 min (flow rate from 0.3 to 0.15 mL min− ). The MetO ratio was calculated in relation to the total pool of Met detected.

4.5. Protein Extraction, Electrophoresis and Western Blot Analysis Ten embryonic axes and ﬁve cotyledons were ground in liquid nitrogen to dry powder, and then the homogenates were incubated in an extraction buﬀer composed of Tris-Cl, glycerol and β-mercaptoethanol together with polyvinylpolypyrrolidone at 4 ◦C for 1 h, with shaking every 15 min. The homogenates were centrifuged for 20 min at 20,000 g at 4 C. The protein concentration × ◦ was measured using the Bradford [105] method. Proteins were separated by SDS-PAGE on 12% polyacrylamide gels, with an equal amount of protein (20 µg) in each lane (Figure S1). The Western blot analysis was performed according to the method described by Wojciechowska et al. [64]. The primary antibodies anti-MsrB1 and anti-MsrB2 [87] were diluted 1:1000 in 5% skimmed milk/PBS. Secondary antibodies conjugated with horseradish peroxidase (HRP, catalog number AS09 602, Agrisera, Sweden) were diluted 1:10,000 in 5% skimmed milk/PBS. Western blot results were analyzed densitometrically in triplicate using the UviBand (UviTec, Cambridge, UK) program of the Fire Reader Gel Documentation System. The density of the band was calculated based on the volume (V) of the band as the sum of all 3D intensities (I) coded on a scale of 256 gray levels. The data show relative units obtained from V = ΣniI and the number of pixels inside the area of the band.

4.6. Activity of NAD(P)H-Dependent Enzymes Activity of NAD(P)H-dependent enzymes was measured according to method describing by Alipour et al. [57] based on the reduction of 5,50-dithiobis(2-nitrobenzoic) acid (DTNB) with NAD(P)H to 2-nitro-5-thiobenzoate. The results of the reaction were measured using an Inﬁnite M200 PRO (TECAN) plate reader and Magellan software.

4.7. Statistical Analyses All experiments were performed with three independent biological replicates. Statistically significant differences were indicated with different letters (ANOVA and Tukey’s test at p > 0.05). Pearson’s correlation coefficient analysis was used to evaluate the relationship between particular parameters. Proportional data were transformed prior to analysis using the arcsine transformation. R statistical software was used to calculate Pearson’s correlation coefficients [106]. The corrplot package was used to construct correlation matrices [107].

5. Conclusions Seed germination has never been investigated in relation to Met redox homeostasis. Norway maple and sycamore seeds representing orthodox and recalcitrant categories, respectively, were found to display distinct behavior, sycamore exhibiting a higher germination speed. Both were highly viable, but also differed in ROS and MetO levels, MsrB abundance and global reducing power. ROS peaks, especially those coinciding with MetO peaks, were assigned as ROS-related events linked to faster Int. J. Mol. Sci. 2020, 21, 9197 16 of 21 germination of sycamore seeds. In sycamore, the negative correlation between MetO and MsrB2 indicates that the reversible MetO posttranslational modification could contribute to the fast germination observed in this species. We suggest that balanced Met redox status is an important feature of sycamore seeds contributing to their high germination speed. Further identification of MsrB targets in germinating seeds would help to decipher their roles in repair mechanisms or signaling pathways during this key developmental stage.

Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/23/9197/s1. Author Contributions: Conceptualization, E.M.K.; funding acquisition and project administration, E.M.K.; investigation, N.W., S.A., E.S., K.B., E.M.K.; data analyses, N.W., S.A., E.M.K.; visualization, N.W., E.M.K.; writing—original draft preparation, N.W., E.M.K.; writing—review and editing, N.W., E.M.K., P.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Science Center (Poland), grant No. 2015/18/E/NZ9/00729. Acknowledgments: This research was supported by the Institute of Dendrology of the Polish Academy of Sciences. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

OH Hydroxyl radicals • ABA Abscisic acid Cys Cysteine DAB 3,3-diaminobenzidine DW Dry weight Gas Gibberellins GAPDH Glyceraldehyde-3-phosphate dehydrogenase Grx Glutaredoxin GSI Germination speed index H2O2 Hydrogen peroxide HRP Horseradish peroxidase Met Methionine MetO Methionine sulfoxide Msrs Methionine sulfoxide reductases NADPH Nicotinamide adenine dinucleotide phosphate O2•− Superoxide anion radicals NBT Nitrotetrazolium blue chloride PPP Pentose phosphate pathway RFU Relative ﬂuorescence units ROS Reactive oxygen species RT Room temperature Trx Thioredoxin WAI Week after imbibition

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